ABSTRACT This application is being submitted in response to NOT-TR-18-027. Osteoarthritis (OA) is a painful disorder that affects over 30 million adults in the US. Total joint replacement (TJR) surgery remains the only long-term treatment option, which remains a treatment of last resort; however, because of the associated risks, cost, and the loss of productivity during recovery and rehabilitation. As pain is the most common symptom of OA, current treatment is primarily focused on pain relief with an escalation of drugs from over the counter non-steroidal anti-inflammatory drugs (NSAIDs) to prescription opioids. Currently, the number of OA patients on opioids continues to increase, despite the significant problems associated with their use, mainly, the publicly recognized opioid crisis. If addiction and overdose were not enough, several lines of evidence suggest that opioids may have direct, detrimental effects on the health of joint tissues. There is thus a critical need for the development of safe and effective methods for the treatment of OA pain. We propose to address this unmet clinical need with the microJoint, an in vitro multi-component microphysiological system derived from human stem cells, developed in the parent grant, for mechanistic analysis of OA-associated pain. We will also use this robust system to investigate the impact of opioids on joint health and OA pain. We hypothesize that a distinct combination of factors released from the OA joint mediate pain and hypersensitivity and hyper- innervation of the joint, and that these mediators are differentially released from the various tissue compartments within the joint. Furthermore, we hypothesize that opioids not only increase the rate of joint degeneration, but also the release of pain producing mediators from the joint. To test these hypotheses, we will first utilize our current microJoint chip setup, which includes bone, cartilage, synovium and adipose tissues, to study the influence of simulated OA-like degeneration on the production of pain-associated mediators, assayed using standard primary cultures of rodent and human nociceptive afferents, in particular, those providing innervation of the knee, as well as induced pluripotent stem cell (iPSC)-derived human sensory neuron progenitors. We will also analyze the level and localization of expression of pain mediators in different joint components. Our goal is to differentially interrogate the relative contribution of any and/or all four of the joint tissue compartments as the source of mediators responsible for OA-associated pain. We will then use the microJoint system to examine the effect of simultaneous and separate opioid exposure of the 4 tissue components on joint health, as assayed on the basis of known markers of joint degeneration. The pain-associated biological activities of the conditioned ?synovial fluid?, under normal or OA conditions, will also be assayed. We believe that this pain-enabled microJoint model, once validated, will allow the generation of OA models with different etiologies and the simultaneous test of tissue health and pain level, and assist the development of personalized OA medications.